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Antibody-Based Targeting of Cell Surface GRP94 Specifically Inhibits
Colorectal cancer (CRC) is one of the leading causes of cancer death
worldwide. Cetuximab, a human/mouse chimeric monoclonal antibody, is
effective in a limited number of CRC patients because of cetuximab
resistance. This study aimed to identify novel therapeutic targets in
cetuximab-resistant CRC in order to improve clinical outcomes. Through
phage display technology, we isolated a fully human antibody strongly
binding to the cetuximab-resistant HCT116 cell surface and identified the
target antigen as glucose-regulated protein 94 (GRP94) using proteomic
analysis. Short interfering RNA-mediated GRP94 knockdown showed that
GRP94 plays a key role in HCT116 cell growth. In vitro functional studies
revealed that the GRP94-blocking antibody we developed strongly inhibits
the growth of various cetuximab-resistant CRC cell lines. We also
demonstrated that GRP94 immunoglobulin G monotherapy significantly
reduces HCT116 cell growth more potently compared to cetuximab,
without severe toxicity in vivo. Therefore, cell surface GRP94 might be a
potential novel therapeutic target in cetuximab-resistant CRC, and
antibody-based targeting of GRP94 might be an effective strategy to
suppress GRP94-expressing cetuximab-resistant CRC.
Figure 1. Graphical abstract
In this study, using phage display technology, we tried to isolate an
antibody strongly binding to the surface of HCT116 cetuximab-resistant
CRC from the human synthetic antibody library. Through proteomic
analyses, we identified the target antigen as glucose-regulated protein
94 (GRP94). By overproducing and purifying a fully human monoclonal
antibody specifically targeting GRP94, we demonstrated that the
antibody targeting of cell surface GRP94 potently reduces the tumor
growth of cetuximab-resistant CRC, without severe toxicity. Our findings
suggested that an antibody-based modulation of the cell surface of
GRP94 might be effective against GRP94-expressing CRC. Therefore,
GRP94 might be a potential novel therapeutic target for antibody
therapy.
Figure 2. Structural features of GRP94 domain
Immunoblot Analysis
Twenty microliters of each elute or 15 µg of cell lysates were resolved by
SDS-PAGE and transferred onto nitrocellulose membranes using a wet
transfer system (GE Healthcare Life Sciences, Piscataway, NJ, USA)
GRP94 or β-actin (loading control) was detected by incubation with 20
µg/mL GRP94 IgG, anti-GRP94
Biotechnology, Dallas, TX, USA), or anti-β-actin monoclonal antibody
(1:3000; Santa Cruz Biotechnology) and then with horseradish peroxidase
(HRP)-conjugated anti-human lambda light chain antibody (1:1000; Bethyl
Laboratories, Montgomery, TX) or goat anti-mouse or rabbit secondary
antibody (1:5000; Santa Cruz Biotechnology). Protein bands were
visualized using SuperSignal West Pico chemiluminescent substrate
(Pierce, Rockford, IL, USA) according to the manufacturer’s instructions.
Enzyme-Linked Immunosorbent Assay (ELISA)
Each well of a 96-well plate was coated with 0.1 μg of recombinant human
GRP94 (rhGRP94) (Sino Biological Inc., Beijing, China), blocked with 3%
(w/v) bovine serum albumin (BSA) in phosphate buffered saline (PBS) for
1 h at 37 °C, and incubated with 20 μg/mL GRP94 IgG for 2 h at room
temperature. Plates were washed with PBS thrice, and 100 µL of 3,3′,5,5′-
tetramethylbenzidine (TMB) substrate solution was added to each well.
Optical densities were measured at 450 nm using a VICTOR X4
microplate reader (PerkinElmer, Waltham, MA, USA).
In Vitro Measurement of CRC Cell Growth
To examine the effect of GRP94 knockdown on HCT116 cell growth, 5 ×
10 HCT116 cells that were transfected with scrambled siRNA or with
3
GRP94 siRNA were seeded into wells of 96-wvitroell plates. To examine
the effect of GRP94 IgG on CRC cell growth in, 5 × 10 HCT116, HT29,
LoVo, HCT-8, or Caco-2 CRC cells were seeded into wells of 96-well
plates in the presence or absence of 100 μg/mL cetuximab or GRP94 IgG.
Cell growth was measured for 60 h using an IncuCyte FLR live content
imaging system (Essen Bioscience, Ann Arbor, MI, USA).
HUVEC Viability Assays
HUVECs (density 5 × 10 ) were placed in 0.1% (w/v) gelatin-coated wells
3
of a 96-well plate and incubated in EGM-2 in the presence or absence of
20 μg/mL GRP94 IgG or 36 μg/mL 5-fluorouracil for 48 h at 37 °C. Cell
viability was determined using the Cell Counting Kit-8 (Dojindo
Laboratories, Rockville, MD, USA) according to the manufacturer’s
instructions. The final optical density was measured at 450 nm using a
spectrophotometer (VICTOR X4, PerkinElmer, Norwalk, CT, USA).
Cetuximab-Resistant Colorectal Cancer Growth
Ji Woong Kim 1# , Yea Bin Cho , Kyun Heo and Sukmook Lee 1*
1
1#
Biopharmaceutical Chemistry Major, School of Applied Chemistry, Kookmin University, Seoul 136-702, Korea
In Vivo Mouse Experiments We monitored morphological changes in HUVECs in the presence
All experiments were performed following protocols approved by or absence of GRP94 IgG by labeling the actin cytoskeleton with
IACUC and in accordance with the guidelines of Hallym rhodamine–phalloidin, which is a fluorescent dye that stains F-actin,
University (Hallym 2017-72). To evaluate the effect of GRP94 and found that GRP94 IgG does not induce significant
IgG on HCT116 tumor growth, BALB/c-nude mice were morphological changes in HUVECs
subcutaneously injected with 3 × 10 HCT116 cells. After 7 days,
6
10 mg/kg GRP94 IgG (n = 7), cetuximab (n = 7), or vehicle (n =
10) was intravenously injected twice weekly. Mice were weighed
and tumor sizes were measured once per week up to day 42.
Statistical Analysis
Data were analyzed using two-tailed Student’s t-tests for
comparison between two groups and one-way analysis of
variance with Bonferroni correction for multiple comparisons in
GraphPad Prism 5.0 (GraphPad Software, La Jolla, CA, USA). Figure 5. Effect of GRP94 IgG on endothelial cell toxicity.
Data are presented as mean ± SEM. p-values < 0.05 were
considered statistically significant. To investigate the effect of GRP94 IgG on cetuximab-resistant CRC cell
growth, we generated an HCT116 xenograft mouse model by
subcutaneously injecting HCT116 cells into nude mice. Then, GRP94
IgG or cetuximab was injected intravenously twice weekly for 7–42 days
after HCT116 cell injection. Tumor sizes and body weights were
monitored during this period.
Figure 3. Characterization of GRP94 IgG
To further confirm the specific binding of GRP94 IgG to endogenous
GRP94 on CRC cells, we performed immunocytochemistry with GRP94
IgG and a commercially available GRP94 polyclonal antibody (positive
control) on HCT116 cells. Similar staining patterns were observed with
both antibodies, further indicating that GRP94 IgG binds specifically to
endogenous GRP94.
Figure 6. In Vivo Efficacy and Toxicity of GRP94 IgG
These results suggested that GRP94 IgG might be effective in
suppressing cetuximab-resistant CRC cell growth, without severe in vivo
toxicity.
Figure 4. Effect of short interfering RNA (siRNA)-mediated GRP94
knockdown on HCT116 cell growth. In summary, cell surface GRP94 is a potential novel therapeutic target
To examine the role of GRP94 in cetuximab-resistant CRC cell growth, in CRC, and antibody targeting of GRP94 might be an effective strategy
we performed a short interfering RNA (siRNA)-mediated knockdown of for suppressing tumor growth in GRP94-mediated CRC. On the basis of
GRP94 in HCT116 cells. First, we confirmed the reduced GRP94 currently available evidence, GRP94 IgG likely binds to GRP94
expression in HCT116 cells using immunoblot analysis. We measured overexpressed on the CRC cell surface within a tumor
the effect of GRP94 knockdown on HCT116 cell growth. GRP94 microenvironment. GRP94 IgG specifically and effectively blocks CRC
knockdown significantly decreased HCT116 cell growth, indicating that cell growth without severe toxicity. In future studies, we will investigate
GRP94 plays an important role in HCT116 cell growth. These results the mechanism of action of GRP94 IgG in more detail, and evaluate the
suggest that GRP94 is a key player in cetuximab-resistant CRC cell in vivo efficacy of GRP94 IgG alone and in combination with other
growth. chemotherapeutic agents against GRP94-expressing CRC.
The cell surface of GRP94 is critical for regulating cetuximab-resistant
CRC cell growth. Further, the antibody targeting of cell surface GRP94
might be an alternative strategy to overcome cetuximab resistance for
antibody therapy. This study was the first study to identify the cell
surface of GRP94 as a novel potential therapeutic target in cetuximab-
antibody (1:200; Santa Cruz resistant CRC, and also to show that the antibody-based modulation of
GRP94 might be, at least in part, helpful in overcoming cetuximab
resistance.
Predicted mode of action of GRP94 IgG
Figure 5. Effect of GRP94 IgG on Cetuximab-Resistant CRC Cell Growth
To determine the effect of GRP94 IgG on cetuximab-resistant CRC cell
growth, we used flow cytometry to determine the binding extent of GRP94
IgG or cetuximab on five CRC cell lines cetuximab-resistant HCT116, HT-
29, LoVo, and HCT-8 cells and cetuximab-sensitive Caco-2 cells. GRP94
IgG strongly bound to the surface of all CRC cell lines, similar to cetuximab.
We also evaluated the inhibitory effect of GRP94 IgG or cetuximab on CRC
3
cell growth by culturing the cells in the presence or absence of GRP94 IgG
or cetuximab. CRC cell growth was monitored in real time live-cell imaging.
GRP94 IgG significantly and more potently inhibited the growth of
cetuximab-resistant HCT116, HT-29, LoVo, and HCT-8 cell lines compared
to cetuximab, which had no effect or a weak effect. In addition, in
cetuximab-sensitive Caco-2 cells, both GRP94 IgG and cetuximab inhibited
cell growth. These results showed that GRP94 has a significant inhibitory
effect on CRC cell growth, suggesting that GRP94 IgG might be a potent
inhibitor of cetuximab-resistant CRC growth in vivo.
This work was supported by grants from the Scripps Korea Antibody
Institute (10TS03) and the Bio & Medical Technology Development
Program of the National Research Foundation funded by the Korean
government (NRF-2019M3E5D5065844).